Medical use of spla2 hydrolysable liposomes

ABSTRACT

The present invention relates to medical use of liposomes, more particular the first medical use of sPLA2 hydrolysable liposomes. Such liposomes may be used for targeted delivery of therapeutic agents to cancerous tissue and in such embodiments; the therapeutic agents are typically small molecule antitumor agents. Other aspects of the inventions relates to methods of reducing the side effects of therapeutic agents, e.g. reducing nephrotoxicity, neurotoxicity and gastrointestinal toxicity of a therapeutic agent. Yet another aspect of the present invention relate to methods of prolonging the therapeutic effect of a therapeutic agent.

FIELD OF THE INVENTION

The present invention relates to liposomal drug delivery systems andtheir use in therapy.

BACKGROUND Liposomes for Drug Delivery

Liposomes are microscopic spheres which were developed as drug deliveryvehicles/systems in the 1980s. The first liposome-based pharmaceuticalswere approved for commercial use in the 1990s.

Liposomes have three distinct compartments that can be used to carryvarious compounds such as drugs: The interior aqueous compartment; thehydrophobic bilayer; and the polar inter-phase of the inner and outerleaflet. Depending on the chemical nature of the compound to beencapsulated it will be localised to either of the compartments.

Liposomes are considered a promising drug delivery system since theypassively target tumor tissue by using the pathophysiologicalcharacteristics of solid tumors such as hyperplasia and increasedvascular permeability but also a defect in lymphatic drainage. Thesefeatures facilitate extravasation of nanoparticles and the liposomes canbe retained in the tissue for longer time due to the enhancedpermeability and retention effect (EPR).

The property of liposomes as drug delivery vehicles is cruciallydependent on their surface charge, permeability, solubility, stabilityetc. which is significantly influenced by the lipids comprised in theliposome composition. In addition, the drug to be encapsulated in theliposome may need further requirements to be considered in preparing astable liposome formulation.

Considerations regarding safety and drug efficacy require that liposomeformulations maintain their properties, i.e. remain stable, from thetime of preparation until administration. Furthermore, it is desirablethat such formulations are intact during the transport in the treatedsubject until they reach the target site where the drug is specificallyreleased.

Therapeutic use of negatively charged liposomes may inducenon-IgE-mediated hypersensitivity reactions seen in patients treatedwith liposomal products. These adverse reactions are thought to be aresult of anaphylatoxin production through complement activation.

Repeated dosing of PEGylated liposomal formulations has in some casesresulted in an Accelerated Blood Clearance (ABC-phenomenon) leading to afast clearance from the bloodstream and corresponding increasedaccumulation in liver and spleen when compared to the first dose. TheABC-phenomenon may cause unintended release of encapsulated compound inorgans having accumulated liposomes. Moreover, the ABC-phenomenon istypically non-desired as it may prevent the liposomes from accumulatingat intended sites.

Various targeting strategies for liposomes have been described, e.g.conjugation to cell specific ligands such as antibodies.

sPLA2 Hydrolysable Liposomes

Another approach has been suggested based upon elevated levels ofsecretory phospholipase A2 (sPLA2) in cancerous tissue and also at sitesof inflammation. The basic idea is that liposomes can be prepared whichare hydrolysable by sPLA2 and that hydrolysis by sPLA2 leads to releaseof the drug encapsulated within the liposome. Moreover, the products ofsPLA2 hydrolysis, a lysolipid and a fatty acid act as permeabilizers ofcell membranes leading to increased cell uptake of the drug. Since sPLA2levels are elevated in the cancerous tissues and at sites ofinflammation, sPLA2 activated liposomes may be used to preferentiallydeliver encapsulated drugs to such sites.

A number of documents have described sPLA2 activated liposomes, buttherapeutic applications have so far not been described.

WO0158910 described sPLA2 activated liposomes comprising prodrugs ofmono-ether lyso-phospholipids. This document also describedencapsulation of additional bioactive compounds. However, no therapeuticuse of the described liposomes was disclosed.

WO0176555 suggested the use of a lipid-based drug delivery system fortreatment of diseases or conditions associated with a localized increasein extracellular sPLA2 in cutaneous or subcutaneous tissue of a mammal,for administration of a prodrug of an ether-lysolipid that is activatedby sPLA2. The system further comprised a so-called edge active compound.This document did not disclose topical application to a mammal such as ahuman. Hence no therapeutic use of the described liposomes wasdisclosed.

WO0176556 suggested the use of a lipid-based drug delivery system fortreatment or prevention of a parasitic infection selected fromLeishmaniasis, Tryponosomiasis, malaria, Entaboeba, Histolyticasis and“Oriental liver fluke chlomorchis sinensis”, wherein the systemcomprised prodrugs in the form of lipid derivatives that are activatedby sPLA2. The liposomes may contain an additional bioactive compound. Noactual treatment of the mentioned infections was demonstrated nor wasthe liposomes administered to a mammal such as a human.

WO06048017 and WO07107161 did also describe sPLA2 activated liposomes,but without any disclosure of medical treatment.

Andresen et al, 2005a (Andresen TL, 2005) discussed triggered activationand release of liposomal prodrugs and drugs in cancer tissue by sPLA2.Among others, the authors disclosed data from an experiment showinginhibition of tumour growth in the MT-3 breast xenograft mouse model.Cisplatin encapsulated in sPLA2 degradable liposomes(DSPC/DSPG/DSPE-PEG2000, no amounts of the individual lipids were given)showed increased inhibition of tumour growth as compared to anequivalent amount of free cisplatin. The authors also noted that in invitro experiments, the sPLA2 degradable liposomes loaded with cisplatinwere more cytotoxic than free cisplatin possibly due to an additivemembrane perturbing effect of the hydrolysis products, lysolipid andfatty acids. This effect might be useful for facilitating transmembranediffusion of cisplatin into intracellular target sites. Whether thiseffect can lead to adverse side effects of sPLA2 activated liposomes wasnot discussed.

Andresen et al, 2005b (Andresen T L J. S., 2005) also disclosed datafrom an experiment showing inhibition of tumour growth in the MT-3breast xenograft mouse model.

Even in view of the references discussed above, it is unclear whethersPLA2 activated liposomes can be used therapeutically. They may e.g. berapidly cleared by the cells of the RES because of their typicallynegative charge. They may also simply be too leaky for therapeutic use.Another very unpredictable parameter is toxicity of the sPLA2 liposomes.As mentioned, the products of sPLA2 mediated hydrolysis, lysolipids andfatty acids, may lead to unintended side effects e.g. throughpermeabilization of cell membranes. Moreover, drug release at unintendedsites may occur if sPLA2 is present at increased levels at sites otherthan in tumours. Such unintended drug release may have detrimentalconsequences and prevent therapeutic use of sPLA2 activated liposomes.Drug release at unintended sites may be caused by unanticipated elevatedsPLA2 levels at such sites.

The therapeutically use of negatively charge liposomes could involvenon-IgE-mediated hypersensitivity reactions seen in patients treatedwith liposomal products. These reactions are thought to be a result ofanaphylatoxin production through complement activation.

Repeated dosing of PEGylated liposomal formulations has in some casesresulted in an Accelerated Blood Clearance (ABC-phenomenon) leading to afast clearance from the bloodstream and corresponding increasedaccumulation in liver and spleen when compared to the first dose. TheABC-phenomenon may course unintended release of encapsulated compound inorgans having accumulated liposomes.

Treatment Using Cisplatin

Free cisplatin formulations have some serious side effects. The mostimportant are listed below:

Nephrotoxicity—The major dose-limiting toxicity of cisplatin isdose-related and cumulative renal insufficiency. The administration ofcis-platin using a 6- to 8-hour infusion with intravenous hydrationhasbeen used to reduce nephrotoxicity. However, renal toxicity still canoccur after utilization of these procedures.

Ototoxicity—Ototoxicity has been observed in up to 31% of patientstreated with a single dose of cisplatin 50 mg/m2, and is manifested bytinnitus and/or hearing loss in the high frequency range (4,000 to 8,000Hz). Ototoxic effects may be related to the peak plasma concentration ofcisplatin.

Hematologic—Myelosuppression occurs in 25% to 30% of patients treatedwith cisplatin. Leukopenia and thrombocytopenia are more pronounced athigher doses (>50 mg/m2). Anemia occurs at approximately the samefrequency as leukopenia and thrombocytopenia

Gastrointestinal—Marked nausea and vomiting occur in almost all patientstreated with cisplatin, and are occasionally so severe that the drugmust be discontinued. Nausea and vomiting usually begin within 1 to 4hours after treatment and last up to 24 hours.

Serum Electrolyte Disturbances—Hypomagnesemia, hypocalcemia,hyponatremia, hypokalemia, and hypophosphatemia have been reported tooccur in patients treated with cisplatin and are probably related torenal tubular damage.

Neurotoxicity—Neurotoxicity is usually characterized by peripheralneuropathies. The neuropathies usually occur after prolonged therapy (4to 7 months); however, neurologic symptoms have been reported to occurafter a single dose.

Hepatotoxicity—Transient elevations of liver enzymes, especially SGOT,as well as bilirubin, have been reported to be associated with cisplatinadministration at the recommended doses.

Thus, treatment using free cisplatin has a number of potential sideeffects and there is a need for cisplatin formulations with a reducedrisk of side effects.

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides sPLAs hydrolysableliposomes for medical use. The sPLA2 hydrolysable liposomes preferablycomprise a therapeutic agent such as a small molecule antitumor agent.

Other aspects of the present invention relates to methods of reducingthe side effects of therapeutic agents, e.g. reducing nephrotoxicity,neurotoxicity and gastrointestinal toxicity of a therapeutic agent.

Yet another aspect of the present invention relate to methods ofprolonging the therapeutic effect of a therapeutic agent.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Efficacy of LiPlaCis towards MT-3 (human breast carcinoma)xenografts. Nude mice with exponentially growing tumors were treatedonce weekly with 4 mg/kg cisplatin or LiPlaCis. The control group wastreated with (saline). The cisplatin and saline-treated groups receivedthree injections whereas the LiPlaCis-treated mice only received twoinjections due to toxicity. See example 2 for details.

FIG. 2. Rats (BrlHan:WIST@Mol(GALAS)) (3 rats/treatment) were injectedwith 3 mg/kg cisplatin or LiPlacis and blood was withdrawn at theindicated time points. After acid digestion, the plasma fraction wasanalyzed for platinum content using ICP-MS. See example 2 for details.

FIGS. 3-7 Pharmacokinetics and biodistribution in nude mice bearing MT-3xenografts. Nude mice (3 mice/timepoint/treatment) with exponentiallygrowing MT-3 tumors were injected with a single dose of cisplatin orLiPlaCis (3 mg/kg). After blood withdrawal, the mice were sacrificed atthe indicated time points and tumors and organs were dissected, washedand snap frozen. After acid digestion, the platinum content was measuredby ICP-MS. See example 4 for details.

FIGS. 8-11 Cisplatin concentration in blood as a function of time afteradministration of LiPlaCis. See example 6 for details.

FIGS. 12-14 Cisplatin concentration in blood as a function ofadministered amount of LiPlaCis. See example 6 for details.

FIG. 15 Summary of phase 1 data. See example 6 for details.

FIG. 16. Detailed phase 1 data. WBC WBC˜white blood cells, ANC˜absoluteneutrophil count, PLT˜platelets, Hgb˜hemoglobin, Nau˜Nausea,Vom˜vomiting, Dia˜diarrhea. See example 6 for details.

DISCLOSURE OF THE INVENTION

The present inventors have carried out in vivo studies with sPLA2hydrolysable liposomes (also herein termed sPLA2 activated liposomes).When sPLA2 hydrolysable liposomes loaded with cisplatin (herein alsotermed LiPlaCis) was administered to tumor mice models, an increasedefficacy as compared to administration of free cisplatin was oftenobserved. However, increased efficacy was also often entailed byincreased toxicity leading to death of mice.

Nonetheless, the present inventors initiated a phase 1 dose escalationtrial of cisplatin encapsulated in sPLA2 hydrolysable liposomes inpatients with advanced or refractory tumors. The primary endpoint of thestudy was safety and tolerability of cisplatin encapsulated in sPLA2hydrolysable liposomes (also termed LiPlaCis).

The main conclusions of the study were:

-   -   LiPlaCis has a tolerable tox profile at clinically relevant        doses.    -   LiPlaCis enables administration of at least the same dose of        cisplatin as administration of free cisplatin, which is        surprising in view of non-clinical data.    -   LiPlaCis reduced nephrotoxicity as compared to administration of        free cisplatin, which is typically dose limiting for cisplatin.    -   LiPlaCis reduced nausea and vomiting as compared to        administration of free cisplatin    -   The MTD (maximum tolerated dose) of LiPlaCis given every 3 weeks        was determined to be above 80 mg per treatment cycle, which is        surprising in view of the MTD predicted from animal experiments.    -   The RD (recommended dose) of LiPlaCis given every 3 weeks was        determined to 80 mg per treatment cycle or higher.    -   LiPlaCis can be administrated without hydration, which is        required for administration of free cisplatin. LiPlaCis can be        administered on an outpatient basis.

In particular, LiPlaCis had good safety and tolerability profilecompared to free cisplatin formulations in terms of nausea, diarrhea,vomiting, anemia, neuropathy, nephrotoxicity and ototoxicity.

Thus, the present invention has made medical use of sPLA2 hydrolysableliposomes available and in its broadest aspect provides a sPLA2hydrolysable liposome for use in therapy, preferably treatment ofhumans.

sPLA2 Hydrolysable Liposomes

sPLA2 hydrolysable liposomes for use in therapy according to the presentinvention are defined in more detail in the following embodiments. Inits broadest embodiment, the term sPLA2 hydrolysable liposomes refer toliposomes that are hydrolysable under physiological conditions,particular in cancerous tissue.

Preferably, the sPLA2 hydrolysable liposomes comprises between 20% and45% (mol/mol) of an anionic lipid. The content of anionic lipid affectsimportant characteristics of the liposome, such as the rate of sPLA2mediated lipid hydrolysis of the liposome and also the immune responsetoward the liposome.

As the content of anionic lipid increases, so does the rate of lipidhydrolysis by sPLA₂ (and the release of drug). It has been demonstratedthat a reasonable rate of hydrolysis can be achieved by an anionic lipidcontent between 20% and 45%. Thus, in one embodiment, the content ofanionic lipid is at least 20%. In another embodiment, the content ofanionic lipid is no more than 45%. In yet another embodiment, theanionic lipid content of the liposome is selected from the groupconsisting of between 20% and 45%, between 25% and 45%, between 28% and42%, between 30% and 40%, between 32% and 38% and between 34% and 36%.

As mentioned, also the immune response toward the liposomes is affectedby the content of anionic lipid. Thus, the clearance rate of theliposome in the body may be reduced by keeping the content of theanionic lipid in the liposome below a certain level and the presentinventors have recognized that the content of anionic lipid in theliposome can be used to strike a balance between hydrolysis rate ofsPLA₂ and clearance by the reticuloendothelial system.

Preferably the anionic lipid is a phospholipid and preferably, thephospholipid is selected from the group consisting of PI (phosphatidylinositol), PS (phosphatidyl serine), DPG (bisphosphatidyl glycerol), PA(phosphatidic acid), PEOH (phosphatidyl alcohol), and PG (phosphatidylglycerol). More preferably, the anionic phospholipid is PG. Preferably,the lipids comprise stearoyl chains. Thus preferably PG is DSPG etc.

Hydrophilic Polymers

In a preferred embodiment, the sPLA2 hydrolysable liposome for use inthe present invention further comprises a hydrophilic polymer selectedfrom the group consisting of PEG [poly(ethylene glycol)], PAcM[poly(N-acryloylmorpholine)], PVP [poly(vinylpyrrolidone)], PLA[poly(lactide)], PG [poly(glycolide)], POZO[poly(2-methyl-2-oxazoline)], PVA [poly(vinyl alcohol)], HPMC(hydroxypropylmethylcellulose), PEO [poly(ethylene oxide)], chitosan[poly(D-glucosamine)], PAA [poly(aminoacid)], polyHEMA[Poly(2-hydroxyethylmethacrylate)] and co-polymers thereof.

Most preferably the polymer is PEG with a molecular weight between 100Da and 10 kDa. Particular preferred are PEG sizes of 2-5 kDa (PEG2000 toPEG5000), and most preferred is PEG2000.

The inclusion of polymers on liposomes is well known to the skilledartisan and can be used to increase the half-life of the liposomes inthe bloodstream, presumably by reducing clearance by thereticuloendothelial system. Moreover, the inclusion of polymers affectssPLA2 hydrolysis.

Preferably, the polymer is conjugated to the head group of phospatidylethanolamine. Another option is conjugation to ceramide (even thoughthis lipid is not hydrolyzable by sPLA₂). When the polymer is conjugatedto phospatidyl ethanolamine, a negative charge is introduced and henceDSPE-PEG is regarded as an anionic lipid (contrary to DSPE which isregarded as a neutral lipid). The polymer-conjugated lipid is preferablypresent at an amount of at least 2%. More preferably, the amount is atleast 5% and no more than 15% (mol/mol). Even more preferably, theamount of polymer-conjugated lipid is at least 3% and no more than 6%.Liposomes containing anionic phospholipids and ≦2.5% DSPE-PEG2000 haveincreased tendency to aggregate in the presence of calcium. This canusually be observed by formation of high viscous gel. Liposomescontaining anionic phospholipids and >7.5% DSPE-PEG2000 causes theliposomes to sediment or phase separate.

Neutrally Charged Lipid Components in the Liposome

Preferably, the liposome to be used in the present invention alsocomprises an uncharged phospholipid selected from the group consistingof zwitterionic phospholipids comprising PC (phosphatidyl choline) andPE (phosphatidylethanolamine). Most preferably, the zwitterionicphospholipid is PC.

In contrast to anionic phospholipid, zwitterionic phospholipid serves asa charge neutral sPLA₂-hydrolyzable lipid component in the liposome. Bycombining zwitterionic- and anionic phospholipid in the same liposome,it is possible to adjust to a desired surface charge density whichcomplies with both sufficiently high sPLA₂ hydrolysis and a lowclearance rate in the blood.

The amount of zwitterionic phospholipid in the liposome is preferablybetween 40% and 75% and more preferably between 50 and 70%.

Preferably, the lipids (anionic lipids, neutral lipids and polymerconjugated lipids) comprise stearoyl chains). Thus preferably PG isDSPG, PE is preferably DSPE etc.

Ether-Phospholipids

Some or all of the phospholipids may be ether-phospholipids.

Thus, they may harbour an ether-bond instead of an ester-bond at thesn-1 position of the glycerol backbone of the phospholipid. When sPLA₂hydrolyze this particular type of phospholipids, mono-etherlyso-phospholipids are produced and these are toxic to e.g. cancercells. I.e. ether phospholipids may be seen as pro-drugs of mono-etherlyso-phospholipids and liposomes of the invention can be used to deliversuch pro-drugs to the sPLA₂-enhanced environment of cancer cells, wherethe pro-drugs are activated by sPLA₂ hydrolysis. Ether-phospholipidshave been described in EP 1254143 and WO 2006/048017, the contents ofwhich are hereby incorporated by reference.

In one embodiment, the sPLA2 activated liposomes as used in the presentinvention does not comprise ether-phospholipids.

Other Pro-Drugs

The moiety released from the lipid by sPLA₂ to create a lysolipid mayalso be a drug. Thus, a liposome may comprise pro-drugs of mono-etherlysolipids, pro-drugs released from the lipid by sPLA₂ and othertherapeutic agents, as further outlined below.

In one embodiment, the sPLA2 activated liposomes as used in the presentinvention does not comprise prodrugs released from the lipid by sPLA2.

Stabilizing Agent

The liposome may be stabilized by the inclusion of cholesterol asmembrane component in the liposome. However, high amounts of cholesterolin the liposome have a negative effect on hydrolysis by PLA₂ andtherefore it is preferred that the liposome comprises no more than 10%cholesterol. Even more preferably, the liposome comprises less than 1%cholesterol, less than 0.1% or does not comprise any cholesterol at all.

The alkyl chain length of the lipids comprising the liposome may beadjusted for optimal PLA₂ hydrolysis rate and minimum leakage ofentrapped compound out of the liposome. Preferably, the alkyl chains areC18 or C16 saturated chains.

The liposomes to be used may be stabilized by exposure to divalentcations.

As described above, the liposomes may comprise pro-drugs of mono-etherlyso-lipids and/or of the moiety released from the lipid by sPLA₂ tocreate the lysolipid.

In a preferred embodiment, the liposomes comprise a bioactive compoundsuch as a therapeutic agent (drug), which is not a pro-drug ofmono-ether lysophospholipid or a mono-ether lysophospholipid. Theliposome may also comprise pro-drugs of mono-ether lysophospholipid anda therapeutic agent. Preferred bioactive compounds are small molecules,peptides, proteins and nucleic acids such as plasmids andoligonucleotides. A preferred class of proteins is antibodies, morepreferably monoclonal antibodies. Preferred oligonucleotides areaptamers, antisense oligonucleotides, microRNAs and siRNAs. A class ofcompounds of particular interest is small molecule antitumour agentssuch as anthracyclin derivatives, cisplatin, oxaliplatin, carboplatin,doxorubicin, paclitaxel, 5-fluoruracil, exisulind, cis-retinoic acid,suldinac sulphide, methotrexate, bleomycin and vincristine. A preferredsubclass of antitumor agents is platinum based antitumor agents;cisplatin, oxaliplatin, picoplatin and carboplatin. Another class ofcompounds of particular interest is antibiotics and antifungals and yetanother class is anti-inflammatory agents such as steroids andnon-stereoids.

The therapeutic agent may be located in the interior aqueouscompartment; the hydrophobic bilayer; or the polar inter-phase of theinner and outer leaflet.

Preferably, the therapeutic agent is encapsulated in the liposome, i.e.present in the interior aqueous compartment.

In another embodiment, the liposome comprises a diagnostic agent. By“diagnostic agent” is meant an agent that supports the localisation ofthe target tissue and/or the diagnosis of the disease and/or condition.Non-limiting examples could be contrast agents, microparticles,radioactive agents, target specific agents such as e.g. agents that bindspecifically to markers associated with the disease and/or condition,etc. It is clear to a skilled person that in some embodiments theinvention relates to a liposome formulation wherein the liposomecomprises at least one drug as well as a diagnostic agent.

Physical-Chemical Characteristics of the Liposomes of the Invention

The liposome can be unilamellar or multilamellar. Most preferably, theliposome is unilamellar. The diameter of the liposome should be between50 and 400 nm, preferably between 80 and 160 nm and most preferablebetween 90 and 120 nm.

Preferably, the Poly Dispersity Index (PDI) of the liposomal formulationof the second aspect of the invention should not exceed 0.2 and morepreferable is 0.10 or less. A PDI value in this range expresses arelatively narrow particle size-distribution in the formulation.

As will be clear from the above, it is preferred that at least one ofthe lipids comprising the liposome is a substrate for sPLA₂ when presentin the liposome.

In one embodiment, the liposome comprises lipids which are hydrolysed bysPLA₂ at the sn-3 position instead of at the sn-2 position. Suchunnatural lipids and liposomes comprising unnatural lipids have beendisclosed in WO 2006/048017, the content of which is hereby incorporatedby reference.

In a most preferred embodiment, the liposomes to be used in the presentinvention comprise 70% DSPC, 25% DSPG and 5% DSPE-PEG.

When the therapeutic agent is cisplatin, the interior of the liposomespreferably comprises 0.9% NaCl and the exterior buffer solutioncomprises 10 mM phosphate buffer at pH 6.5, 1 mM NaCl and 10% sucrose.

Medical Use

In a preferred embodiment, the sPLA2 hydrolysable liposome isadministered by injection (parenteral administration) e.g. thesubcutaneous, intramuscular, intra-peritoneal, intravenous, andintrathecal routes. A preferred route is intravenous administration inform of bolus injection or infusion.

As described above, the liposome may comprise various therapeuticagents. However, preferred agents are small molecule anti tumour agents(herein also termed antineoplastic agents, cytotoxic drugs or cytostaticdrugs). Cisplatin is one of these compounds and the demonstration thatcisplatin encapsulated in a sPLA2 hydrolysable liposome can be usedtherapeutically, argues that other antineoplastic agents encapsulated insPLA2 hydrolysable liposomes can also be used therapeutically, i.e. theywill not be released from sPLA2 hydrolysable liposomes at unintendedsites at a concentration which would be detrimental to the therapeuticuse of sPLA2 hydrolysable liposomes encapsulating antineoplastic agents.

In a preferred embodiment, the administration of sPLA2 hydrolysableliposomes comprising a therapeutic agent enables administration of areduced dose of the therapeutic agent as compared to administration ofthe free therapeutic agent. This is possible for several reasons. First,liposomal encapsulation of the therapeutic agent prolongs the half-lifeof the agent. Second, the targeting effect of sPLA2 hydrolysis leads toan increased concentration of the free therapeutic agent at sites ofincreased sPLA2 levels, e.g. at tumours.

In another preferred embodiment, the administration of sPLA2hydrolysable liposomes comprising a therapeutic agent enablesadministration of an increased dose of therapeutic agent as compared toadministration of the free therapeutic agent. This is possible becauseof the targeting effect of sPLA2 hydrolysable liposomes and can e.g. beseen by reduced nephrotoxicity of cisplatin encapsulated in sPLA2hydrolysable liposomes as compared to free cisplatin.

LiPlaCis have been studied in a number of non-clinical toxicologystudies in rats and mice. The overall purpose of these studies was todetermine both the single dose and multiple dose Maximum Tolerated Dose(MTD) in the two species. These studies were conducted according to GoodLaboratory Practice (GLP). In these studies the two species was found tobe equally sensitive to LiPlaCis and by applying FDA rules (Reference:Guidance for Industry. Estimating the Maximum Safe Starting Dose inInitial Clinical Trials for Therapeutics in Adult Healthy Volunteers.FDA, July 2005), the human equivalent maximum tolerated dose ispredicted to be 30 mg per treatment cycle. Thus, a human MTD of 80 mg ormore per treatment cycle is surprising.

Preferred doses of encapsulated cisplatin are between 80 mg and 120 mgper treatment cycle with a 3 week interval, between 120 and 160 mg pertreatment cycle with a 3 week interval, between 160 mg and 200 mg pertreatment cycle with a 3 week interval, between 200 mg and 240 mg pertreatment cycle with a 3 week interval and between 240 mg and 300 mg pertreatment cycle with a 3 week interval.

The time between administrations of therapeutic agent may also beadjusted in line with the discussion of reduced/increased doses oftherapeutic agent. Thus, in one embodiment, the time betweenadministrations of therapeutic agent is prolonged as compared to thetime between administrations of the free therapeutic agent. In anotherembodiment, the time between administrations of therapeutic agent isreduced as compared to the time between administrations of the freetherapeutic agent. When the therapeutic agent is cisplatin, the timebetween administrations may e.g. be more than 3 weeks or less than 3weeks.

Preferably, the disease to be treated according the invention is canceror inflammation, preferably cancer.

Method of Treatment

A second aspect of the present invention is a method of treatmentcomprising administering an effective amount of an sPLA2 hydrolysableliposome as described in the first aspect of the invention to a patientin need thereof. Specific embodiments of this aspect will be apparentfrom the first aspect of the invention.

Method of Reducing Nephrotoxicity

A third aspect of the invention is a method of reducing thenephrotoxicity of a therapeutic agent, said method comprisingencapsulating the therapeutic agent in a sPLA2 hydrolysable liposome.Preferably, the therapeutic agent is an antineoplastic agent such ascisplatin and preferably, the therapeutic agent is administered to apatient in need thereof. Other embodiments will be apparent from thefirst aspect of the invention.

Method of Reducing Neurotoxicity

A forth aspect of the invention is a method of reducing theneurotoxicity of a therapeutic agent, said method comprisingencapsulating the therapeutic agent in a sPLA2 hydrolysable liposome.Preferably, the therapeutic agent is an antineoplastic agent such ascisplatin and preferably, the therapeutic agent is administered to apatient in need thereof. Other embodiments will be apparent from thefirst aspect of the invention.

Method of Reducing Gastrointestinal Toxicity

A fifth aspect of the invention is a method of reducing thegastrointestinal toxicity of a therapeutic agent, said method comprisingencapsulating the therapeutic agent in a sPLA2 hydrolysable liposome.Preferably, the therapeutic agent is an antineoplastic agent such ascisplatin and preferably, the therapeutic agent is administered to apatient in need thereof. Other embodiments will be apparent from thefirst aspect of the invention

Method of Prolonging the Therapeutic Effect

A sixth aspect of the invention is a method of prolonging thetherapeutic effect of a therapeutic agent, said method comprisingencapsulating the therapeutic agent in a sPLA2 hydrolysable liposome.Preferably, the therapeutic agent is an antineoplastic agent such ascisplatin and preferably, the therapeutic agent is administered to apatient in need thereof. Other embodiments will be apparent from thefirst aspect of the invention. References

REFERENCES

-   Andresen T L, J. S. (2005). Advanced strategies in liposomal cancer    therapy: problems and prospects of active and tumor specific drug    release. Prog Lipid Res., January; 44(1):68-97. Epub 2005 January    22.-   Andresen T L, J. S. (2005). Triggered activation and release of    liposomal prodrugs and drugs in cancer tissue by secretory    phospholipase A2. Curr Drug Deliv, October; 2(4):353-62.

EXAMPLES Example 1 Preparation of sPLA2 Liposomes (LiPlaCis)

A lipid intermediate is prepared by spray-drying the following a mixtureof phospholipids (70/25/5 mol % DSPC/DSPG/DSPE-PEG2000). The lipids aredissolved in methanol and chloroform. The lipid intermediate is hydratedin an aqueous solution of the anti-cancer drug with agitation. At thisstep the liposomes are formed but they have a broad size distributionand is a mixture of single-layer and multiple-layer liposomes. In orderto get a product with a narrow size distribution and mono-layerliposomes the hydration mixture is extruded by passing it throughpoly-carbonate filters of appropriate pore sizes. To removeun-encapsulated anti-cancer drug the mixture is purified. A number oftechniques are available e.g. dialysis, gel-filfration andultra-filtration. For preparations ranging from a few liters and aboveultra-filtration is the preferred method. Preparations intended forparenteral administration must be sterilized e.g. by sterile-filtration.

Example 2 Efficacy in Mice Methods

NMRI nude female mice (6-8 weeks) were inoculated subcutaneously intothe left flank with 1*10⁷ cells of the human breast carcinoma cell lineMT-3. Only mice carrying exponentially growing tumors were selected forthe study. Treatment started when tumors had reached a size of 70-80mm3. Animals received one dose (4 mg/kg cisplatin (Platinol), LiPlaCisor saline) weekly with intra-venous injections into the tail veinstarting on day 13 after tumor transplant. Tumor growth was assessedthree times a week by measuring two perpendicular diameters and tumorgrowth was normalized for differing starting sizes by calculatingrelative tumor volume. Body weight was measured three times a week.Blood samples were taken four days after the first injection to estimatewhite blood cells and thrombocytes by Coulter counter.

Results:

LiPlaCis was compared with cisplatin and saline in an efficacy studyusing MT-3 breast xenografts on nude mice. Cisplatin and LiPlaCis weregiven at a dose of 4 mg/kg weekly. Because of toxicity, only twoinjections of LiPlaCis were administrated compared to three forcisplatin and saline. LiPlaCis inhibited tumor growth significantlybetter than free cisplatin (FIG. 1). The effect was apparent a weekafter of the first dosing and lasted till the experiment was terminatedbecause of large tumors in the control group. One mouse died in theLiPlaCis-treated group.

TABLE 1 Experimental parameters and toxicity: Treatment Dose tox BWC (%)Optimum T/C WBC d 17 Thromb. d 17 Group mice Subst. (days) (mg/kg/inj.)deaths(d) d 13-24 (%) [at day] (10{circumflex over ( )}6/ml)(10{circumflex over ( )}6/ml) A 10 Saline 13, 20, 27 0  −2  9.7 +/− 1.21185 +/− 89 B 10 Cisplatin 13, 20, 27 4 0  −6 71 [31] 10.6 +/− 1.5 1201+/− 117 C 10 LiPlaCis 13, 20 4 1 (26) −14 31 [26]*+ 11.2 +/− 2.7 1058+/− 183 BWC: Body Weight Count, difference in percentage compared to theweight before treatment. Optimum T/C: Quote of treated tumors dividedwith control tumors. WBC: White Blood Cells Thromb: ThrombocytesLiPlaCis appears to lead to higher bioavailability of cisplatin andinduce more potent anti-tumor efficacy but has more intense side effectsthan free cisplatin including body weight loss and thrombopenia.

Example 3 Pharmacokinetics in Rats Methods:

Rats (BrlHan:WIST@Mol(GALAS)) were injected with 3 mg/kg cisplatin orLiPlaCis and blood was collected into heparinised tubes (Microvette CB300 Sarsted). Samples were taken from 10 minutes up to 72 h. A bloodvolume of 250 μl was taken from each sampling point and immediatelyplaced in an ice-bath and centrifuged (3000×g; 5 min) to obtain theplasma fraction. The plasma-containing tubes were frozen until shipmentand subsequent digestion in HCl/HNO3/H2O2 (60/5/35 vol %) beforeplatinum analysis using ICP-MS.

Results and Conclusion:

The experiment revealed that LiPlaCis a long-circulating liposomal formof cisplatin with a T½ of about 20-23 h compared to the 15 minutes forfree cisplatin. The area under the curve (AUC) for LiPlaCis was at least50 times that of cisplatin. See FIG. 2.

Example 4 PK/BD in Nude Mice Methods:

Nude BALB/c female mice (6-8 weeks) were inoculated subcutaneously intothe left and right flank with 1×10⁷ cells of the human breast carcinomacell line MT-3. Only mice carrying exponentially growing tumors wereselected for the study. The single dose was given when the tumors hadreached a size of at least 300 mm3. Animals received 3 mg/kg cisplatin(Platinol) or LiPlaCis) by tail vein injection. The time points were 1,24, 48, 72 and 168 h. Blood samples (500 μl) was taken immediatelybefore sacrifice and transferred to heparinised tubes (Microvette CB 300Sarsted), centrifuged and frozen. Post mortem, the tumors, organs andtissues (kidneys, liver, quadriceps muscle on the hind limb and spleen)were dissected, washed in saline and snap frozen. To determine platinumconcentrations in plasma and tumors/tissues, the samples were digestedin HCl/HNO3/H2O2 (60/5/35 vol %) and subjected to ICP-MS.

Results:

Platinum analysis in plasma showed that LiPlaCis was present at highconcentrations in serum and the effect was lasting for at least a week.The levels of LiPlaCis in serum were at any time-point more than anorder of magnitude higher than free cisplatin. LiPlaCis also accumulatedin tumors with a maximum of about 4 μg/mg tumor mass compared to about 1μg/mg tumor for free cisplatin. There were no significant differences inplatinum accumulation in the liver. There was a moderate accumulation ofLiPlaCis in the kidneys whereas the highest levels of platinum could bemeasured in the spleen from LiPlaCis-treated animals. See FIGS. 3-7.

Conclusion PK/BD:

LiPlaCis long-circulating liposomal form of cisplatin. LiPlaCisaccumulates in tumors and also in kidneys and spleen. Cisplatin can bereleased from LiPlaCis in the tumor microenvironment

Example 5 Nephrotoxicity

In humans receiving cisplatin therapeutically, a major side effect andthe dose limiting toxicity of cisplatin is nephrotoxicity. In this studythe nephrotoxicity of cisplatin was compared with that of LiPlaCis inthe rat.

Methods

Groups of five male and five female Wistar rats, 6-7 weeks old and witha body weight of 145-175 g, were given an intravenous injection ofeither 3 mg/kg of Cisplatin or 3 mg/kg of LiPlaCis.

Two days after the injection, two males and two females from each groupwere sacrificed, 7 days after the injection, one male and one femalefrom each group were sacrificed, and 14 days after the injection theremaining two males and two females from each group were sacrificed. Theanimals were subjected to macroscopic pathology and absolute andrelative kidney weights were recorded. Histopathology was performed onkidneys, urinary bladder and spleen from all animals.

Results and Conclusion

At necropsy, the kidney weights were generally higher after treatmentwith LiPlaCis, and the histopathological examination showed thattreatment with LiPlaCis clearly reduced the severity of renaldegenerative changes in the form of multifocal tubular basophilia/debrisand diffuse tubular vacuolation and dilation. Treatment with LiPlaCispresumably also caused a lower incidence of decreased cellular densityof the white pulp/periarterial sheath compared with Cisplatin. Inconclusion, LiPlaCis clearly reduced the nephrotoxicity of Cisplatin inrats.

Example 6 Phase I Dose-Escalating Study to Evaluate the Safety andTolerability of LiPlaCis (Liposomal Cisplatin Formulation) in Patientswith Advanced or Refractory Tumors Study Synopsis Primary Objective:

-   -   1. To evaluate the safety and tolerability of LiPlaCis given        every 3 weeks    -   2. To determine the maximum tolerated dose (MTD) and the        recommended dose (RD) of LiPlaCis given every 3 weeks

Secondary Objectives:

-   -   3. To evaluate the pharmacokinetics (PK) of LiPlaCis given every        3 weeks    -   4. To evaluate the therapeutic efficacy of LiPlaCis given every        3 weeks

Study Design:

Open label, non-randomised dose escalation study

Study Population:

Subjects with a solid tumor not amenable to standard treatment

Number of Patients:

The precise number of patients cannot be defined, as this is dependenton the observed toxicity. Cohorts of 3 to 6 patients will be treated ateach dose level until MTD is reached. It is anticipated that 30 patientscould be needed to assess MTD.

Eligibility Inclusion Criteria:

-   -   1. Histological or cytological documented locally advanced or        metastatic solid tumor refractory to standard therapy or for        which no curative therapy exists.    -   2. Be ≧18 years of age.    -   3. Have a life expectancy 3 months.    -   4. Have an ECOG performance status of 0-2.    -   5. Have recovered to grade 1 or less from acute toxicities of        prior treatment:    -   6. ≧6 months must have elapsed since patient received cisplatin.    -   7. ≧4 weeks must have elapsed since patient received any        investigational medicinal product.    -   8. ≧4 weeks must have elapsed since patient received any        radiotherapy, or treatment with cytotoxic or biologic agents (≧6        weeks for mitomycin or nitrosoureas). No hormonal treatment is        allowed except treatment with corticosteroids at physiological        dose and hormonal treatment with LHRH agonists for prostate        cancer.    -   9. ≧2 weeks must have elapsed since any prior surgery, blood        transfusions or therapy with GM-CSF. However, current use of        erythropoietin will be permitted.    -   10. Be in adequate condition as evidenced by the following        clinical laboratory values:        -   a. Absolute neutrophil count (ANC)≧1.5×109/L        -   b. Haemoglobin is at least 9 g/dl (5.6 mmol/L)        -   c. Platelets≧100×109/L        -   d. Alanine aminotransferase (ALT) and aspartate            aminotransferase (AST)≦2.5×ULN; in case of known liver            metastases ALT and AST ≦5×ULN        -   e. Serum bilirubin ≦1.5 ULN        -   f. Alkaline phosphatase ≦2.5×ULN        -   g. Creatinine and blood urea within normal limits, unless            creatinine clearance is within normal limits (≧60 mL/min            calculated according to Cockcroft-Gault formula) (see            appendix 1)    -   11. Patients (male and female) must be willing to practice an        effective method of birth control during the study.    -   12. Patient or legal representative must understand the        investigational nature of this study and sign an independent        ethical committee (IEC) approved written informed consent form        prior to treatment.

Exclusion Criteria are the Following:

-   -   1. Active uncontrolled bleeding or bleeding diathesis (e.g.,        active peptic ulcer disease).    -   2. Any active infection requiring parenteral or oral antibiotic        treatment.    -   3. Known infection with human immunodeficiency virus (HIV) or        hepatitis virus.    -   4. Active heart disease including myocardial infarction or        congestive heart failure within the previous 6 months,        symptomatic coronary artery disease, or symptomatic arrhythmias        currently requiring medication.    -   5. Known or suspected active central nervous system (CNS)        metastasis. (Patients stable 8 weeks after completion of        treatment for CNS metastasis are eligible.)    -   6. Autoimmune disease.    -   7. Impending or symptomatic spinal cord compression or        carcinomatous meningitis.    -   8. Having pre-existing neuropathy, i.e., Grade >1 neuromotor or        neurosensory toxicity (as defined by National Cancer Institute        Common Toxicity Criteria for Adverse Events (NCI CTCAE) v3.0),        except for abnormalities due to cancer.    -   9. Having known hypersensitivity to cisplatin or liposomes.    -   10. Requiring immediate palliative treatment of any kind        including surgery and/or radiotherapy.    -   11. Female patients who are pregnant or breast-feeding        (pregnancy test with a positive result before study entry).    -   12. Unwilling or unable to follow protocol requirements.

Study Procedures:

Adverse events (AEs): From signing informed consent of study drug until30 days after receiving the last dose of study drug. Related AEs, incl.serious AEs, are followed until returned to baseline or grade tograde 1. Physical examination, vital signs, Performance status, Bloodchemistry, Urinalysis: baseline and weekly in each cycle.

Haematology: baseline, bi-weekly in cycle 1 and weekly in other cycles.

-   -   Pharmacokinetic (PK) sampling: blood and urine samples should be        obtained for PK evaluation. Please see section 6.4 Clinical        Pharmacology Procedures.

Tumor assessments: baseline and every 3 cycles.

Study Assessments:

Safety, as determined by physical examinations, laboratory toxicity, andthe incidence and severity of adverse events.

Safety assessments: NCI Common Technology Criteria for Adverse Events(CTCAE) version 3.0, laboratory evaluations (biochemistry, haematology),vital signs, physical examination including neurological examination,ECOG performance status and body weight.

Maximum tolerated dose of LiPlaCis as determined by dose-limitingtoxicities (DLTs) and the recommended dose.

Clinical response rate will be determined by radiographic criteria usingRECIST.

Efficacy assessments (if applicable): overall tumor response accordingto RECIST (CR, PR, SD or PD).

Rationale for the Study Rationale for Selecting Dose and Schedule:

The human starting dose is determined by using the approach suggested byFDA (Reference: Guidance for Industry. Estimating the Maximum SafeStarting Dose in Initial Clinical Trials for Therapeutics in AdultHealthy Volunteers. FDA, July 2005). Rat MTD is 3 mg/kg and theconversion factor between rat and human is 6.3 according to FDA'sguideline. This gives a human equivalent dose (HED) of 0.5 mg/kg.

Mouse MTD is 6 mg/kg and the conversion factor between mouse and humanis 12.3 according to FDA's guideline. This gives a human equivalent dose(HED) of 0.5 mg/kg.

Thus, when the rat and mouse MTD's are converted into human equivalentdoses is evident that these two species have the same sensitivity whenexposed to LiPlaCis.

The reference human body weight is according to the guideline 60 kgwhich correspond to a dose of 30 mg per patient (0.5 mg/kg*60 kg=30 mg).A safety factor of 3 is applied resulting in a starting dose of 10 mgper patient. This dose represents 1/10 of the recommended lowest dose ofplain cisplatin products (50 to 100 mg/m2 when administered as a singledose every 3-4 weeks) assumed that a normal person's body surface areais 2 m2.

A higher safety factor is frequently used (often 10) but the findings inthe non-clinical studies suggest that the toxicity of LiPlaCisdetermined by the intrinsic toxicity of cisplatin and that LiPlaCis lesstoxic compared to plain cisplatin. Starting at 10 mg per subject shouldthen be well on the safe side. Further it should be noted that LiPlaCisnot a completely new drug, but a new formulation of a very well knownand widely used drug.

Rationale for Schedule and Route of Administration:

In previous clinical phase I and II studies of liposomal cisplatinformulations—e.g. SPI-077 and Lipoplatin developed by ALZA Corp. andRegulon Inc., respectively—the drug product has mainly been administeredevery three weeks and the median number of cycles given has been between2 and 4. In some of these studies, patients were to receive a total of 6cycles.

To ensure that patients can be treated optimally in terms of safety andpotential efficacy, LiPlaCis will be administered every 3 weeks for upto 3 cycles or more if the patient benefits from further cycles in theopinion of the investigator.

LiPlaCis will be administered intravenously by infusion as conventionalcisplatin.

Study Description

The study is an open label, dose-escalating, non-randomised phase Istudy of LiPlaCis in patients with advanced cancer.

LiPlaCis will be administered every 3 weeks for up to 3 cycles or moreif the patient benefits from the treatment upon the investigator'sjudgement and if there is no evidence of progressive disease orunacceptable toxicity. Post-trial access to other care must be evaluatedwhen patients enter the trial.

LiPlaCis will be administered with increases of 20 to 100% from theprevious dose level.

The number of levels needed to reach MTD is unknown. The dose escalationof to 100% will be made based on toxicity and pharmacokinetics afterdiscussion between the investigators and the sponsor.

A clinical (telephone) conference will be organized once the lastpatient in the respective cohort has completed the first cycle todiscuss dose-escalation. The same panel of investigators in discussionwith the sponsor (LiPlasome Pharma) will decide on the MTD and therecommended dose (RD) to be used in future phase II studies of LiPlaCis.

The MTD will be the regimen with two or more patients with DLT in acohort of 3 or 6 patients. Following completion of all cohorts and afterthe MTD has been defined; a clinical conference will be organized toreview the outcomes of the patients to decide on the next dose, and todetermine the RD for LiPlaCis. The RD will normally be the dose levelbelow MTD (MTD-1). RD will be the dose at which no more than 1 out ofthe 6 patients experience DLT in first cycle.

Three patients will be enrolled per dose level and each cohort ofpatients will receive LiPlaCis every 3 weeks to a total of three cyclesor more or until disease progression or unacceptable toxicity occurs(please see definition of dose-liming toxicity in section 6.5). Percohort/dose level the second and third patient can be enteredsimultaneously after evaluation of the first week of the 1st cycle ofthe first patient in that cohort.

The duration of infusion will be 1 hour and could be changed to 3 hoursin case adverse events—e.g. infusion reactions—necessitate a longerduration or a temporary discontinuation of infusion.

If a dose-limiting toxicity (DLT) occurs in one of the three patientswithin one cohort, then three additional patients will be treated atthat level. If a DLT occurs in ⅔ or 2/6 patients, the next lower doselevel will be expanded to at least 6 patients. The last patients of acohort will be observed for 3 weeks before accrual to the next higherdose level might start.

Patients will be replaced within a cohort when they go off study within3 weeks for other reason than toxicity.

The last patient at a dose level should be observed for at least 3 weeksbefore the first patient at the subsequent dose level can be treated.

Antiemetics:

Initially, the study treatment will start without the use ofprophylactic anti-emetics. Once two patients experience nausea and/orvomiting grade 2 or more, prophylactic use of the following anti-emeticswill be introduced for the patient in question and the remainingpatients.

Step 1: 5-HT3 antagonist (e.g. granisetron, ondansetron)

Step 2: Day 1: granisetron 1 mg iv and dexamethason 10 mg iv, Day2-4:dexamethason 6 mg per os

Step 3: Day 1: aprepitant 125 mg per os, granisetron 1 mg iv,dexamethason 10 mg iv; Day2-3: prepitant 80 mg per os, dexamethason 6 mgper os; Day 4: dexamethason 6 mg per os.

If a patient experiences nausea and/or vomiting of grade 2 or more,therapeutic anti-emetics may be administered including Step 0:metoclopramide. At re-treatment this patient may receive prophylacticanti-emetics at investigators decision. The anti-emetics will beadministered in accordance with procedures at Erasmus MC and LUMC.

Hydration:

Hydration will not be used routinely.

However, if nephrotoxicity is observed in a patient, both pre- andpost-hydration will be introduced for the remaining cycles of thispatient Hydration will consist of 1000 mL glucose 2.5%/NaCl 0.45% over 4hours prior to treatment and 3000 mL glucose 2.5%/NaCl 0.45% over 8hours post treatment.

In accordance with the definition of MTD in case nephrotoxicity shouldbe observed in two or more patients in a cohort of 3 or 6 patients pre-and post-hydration will be introduced for the remaining cycles of theremaining patients.

However in case nephrotoxicity is observed in different patient overdifferent cohorts this might also be a reason to start with theintroduction of additional hydration. This will be decided during doseescalation teleconferences with the investigators and the sponsor.

Study Population

The targeted population for this study are patients with histologicallyor cytologically documented locally advanced or metastatic solid tumorrefractory to standard therapy or for which no curative therapy exists.

Number of Patients

The precise number of patients cannot be defined, as this is dependenton the observed toxicity. Cohorts of 3 to 6 patients will be treated ineach cohort until the MTD and the recommended dose for phase II studiesof LiPlaCis determined. It is expected that up to 30 evaluable patientscould enter the study to meet the key objectives of the study. However,more patients will be enrolled if required to do so.

Prior to inclusion, the patients must give written informed consent forthis study and must meet all the selection criteria listed in section3.3. Patients who sign an informed consent but fail to meet theinclusion and/or exclusion criteria are defined as screen failures. Forall patients who have consented, the investigator is to maintain ascreening log that documents the screening number, patient initials, and(if applicable) reason(s) for screen failure. A copy of the log shouldbe retained in the investigator's study file.

Results from Phase 1 Study

PK:

Pharmacokinetic data confirm that LiPlaCis a long circulatoryformulation of cisplatin. The following is observed:

-   -   The observed T½ is 78 hours, which is to be compared with        cisplatins T½ of less than one hour. See FIGS. 8-11.    -   The pharmacokinetic profile is linear both in terms of Cmax and        AUC. See FIGS. 12-14.    -   Urinary excretion is significantly altered compared to        cisplatin. Urine is collected from 0 to 96 hours and excretion        is between 0 and 20% of the administered dose. Cisplatin urinary        excretion is above 90% within 3 hours.

Tox:

LiPlaCis administered in doses up to 120 mg per treatment cycle shows nosign of nephrotoxicity, ototoxicity and neurotixicity. Further,gastrointestinal toxicity in form of nausea and vomiting have not beenreported in patients receiving LiPlaCis. See FIGS. 15 and 16.

1. A sPLA2 hydrolysable liposome for use as a medicament of humans. 2.The liposome of claim 1, wherein the content of anionic lipid is between20% and 45%, the content of polymer conjugated lipid is between 3% and6% and the content of neutral lipids is between 40% and 75%.
 3. Theliposome of claim 2, wherein the anionic lipid is DSPG, the neutrallipid is DSPC and the polymer conjugated lipid is DSPE-PEG.
 4. Theliposome of claim 1, wherein the liposome is administered intravenousadministration in form of bolus injection or infusion.
 5. The liposomeof claim 1, wherein the liposome comprises a therapeutic agent.
 6. Theliposome of claim 1, wherein the therapeutic agent is a small moleculeantitumour agent is selected from the group consisting of: anthracyclinderivatives, cisplatin, oxaliplatin, carboplatin, picoplatin,doxorubicin, paclitaxel, 5-fluoruracil, exisulind, cis-retinoic acid,suldinac sulphide, methotrexate, bleomycin and vincristine.
 7. Theliposome of claim 1, wherein the small molecule antitumor agent is aplatinum based antitumor agent selected from the group consisting ofcisplatin, oxaliplatin, picoplatin and carboplatin.
 8. The liposome ofclaim 1, wherein the dose of the therapeutic agent is reduced ascompared to the standard dose of the free therapeutic agent.
 9. Theliposome of claim 1, wherein the dose of the therapeutic agent isincreased as compared to the standard dose of the free therapeuticagent.
 10. The liposome of claim 1, wherein liposome is administered atprolonged intervals as compared to the standard dose regiment of thefree compound.